Treatment of inflammatory skin disorders

ABSTRACT

The invention relates to methods of using an effective amount of activated protein C (APC) to treat an individual for a skin disorder characterised by the presence of hyperproliferative keratinocytes.

FIELD OF THE INVENTION

The invention relates to skin disorders characterised byhyperproliferation of keratinocytes, including but not limited topsoriasis and to treatment of same.

BACKGROUND OF THE INVENTION

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

Psoriasis, the most prominent autoimmune disease, is both chronic andrelapsing in nature. It is characterized by impaired barrier function,hyperproliferation of epidermal keratinocytes and pronouncedinfiltration of inflammatory cells.

Recent data showed that although inflammatory T cells are integral tothe disease, the keratinocyte is the key driver of pathogenicinflammation in psoriasis, through integrating responses to interleukin(IL)-1, IL-6, IL-8, IL-17, interferon (INF)-γ, and TNF-α (1,2) and theactivation of nuclear factor NF-κB, a signaling molecule that maintainsimmune homeostasis of epidermal keratinocytes (3,4).

There is no cure for psoriasis. Topical medications, phototherapy,traditional systemic agents, and biologics only offers options formanagement of its symptoms. A combination of agents is frequently neededfor moderate-to-severe cases and positive long-term outcomes requiremedication adherence (5).

Typically, localized disease (10% body surface area) is managed withtopical medications while larger areas require phototherapy or systemictherapy. Disease localized to the palm and soles is often treatedsystemically, since skin thickness in this area is not amenable totopical therapy.

Topical treatment agents include corticosteroids such as clobetasol, anda vitamin D analogue such as calcitriol or calcipotriene (6,7). Thecorticosteroid works by decreasing inflammation, suppressing mitoticactivity, and causing vasoconstriction in the targeted area. Themechanism of vitamin D analogues action is not fully understood, but itappears to regulate keratinocyte proliferation and differentiation (6).Thus components of both treatments include inhibition of cellproliferation, although this is non specific.

Topical corticosteroids can cause skin atrophy, irritation, and drynessin the affected area. Vitamin D analogues are generally safe but cancause irritation while there is a risk of hypercalcemia if used in verylarge doses (8).

With extensive psoriatic disease, phototherapy or systemic treatmentsare used. The first-line treatment for extensive psoriasis is throughultraviolet light, such as UVB and UVA. However, this treatment isassociated with a greater risk of burns and skin cancer, particularly inpatients with lighter skin color. Low-dose methotrexate can also be usedif UV light therapy proves ineffective. Methotrexate is reasonablyeffective at controlling psoriasis, but is severely limited by serioustoxic effects.

With the increasing knowledge of the immune nature of the disease,biological agents that target T lymphocytes, TNF-α, IL-12, and IL-23have been successfully utilized in moderate-to-severe psoriasis (8).Three commonly used TNF inhibitors include Enbrel (etanercept), Humira(adalimumab), and Remicade (infliximab). Ustekinumab, a new agent thattargets IL-12 and IL-23 was approved for marketing in 2009, offeringsimilar efficacy and safety profiles to anti-TNF agents (9).

Biological agents offer considerable advantages over previouslyavailable systemic therapies, however, clinical trials of these drugdemonstrated that the activity of such compounds is accompanied in thelong term by a number of disadvantages (10-11). Firstly, in patientswith latent mycobacterium tuberculosis (TB) infection, active TB maydevelop soon after the initiation of treatment with infliximab. Inaddition, patients on TNF inhibitors are at increased risk ofopportunistic fungal infections, such as pulmonary and disseminatedhistoplasmosis, coccidioidomycosis, and blastomycosis. Secondly, adose-dependent increased risk of malignancies including lymphoma andskin cancer applies to some treatments. Thirdly, a proportion ofpatients do not respond to the agents, or fail to maintain initialresponse. The availability of the biological agent is also limited byits high economical cost.

Activated protein C (APC) induces the gene expresion of the antiapoptotic human genes bcl-2 and MIHB. Based on this anti-apoptoticfunction, APC has been mentioned for minimisation of apoptosis in a widerange of conditions where apoptosis is of concern, including a largerange of organ specific and non specific inflammatory diseases,autoimmune diseases, neoplasia and infectious disease. Notably the useof APC is not suggested to treat all symptoms of disease in thesecondition, merely to minimise apoptosis (see WO2001/072328).

Further, many in the field are of the view that the anti-apoptoticeffects, such as suggested in WO2001/072328, should make APC unsuitablefor treatment of disorders where there is dysfunctional regulation ofcell proliferation, especially disorders involving keratinocytehyperproliferation. In particular, it is known that, apart from theattenuation of calcium-induced cell death via prevention of cellapoptosis by APC, APC also has a potent stimulatory effect onkeratinocyte proliferation, and APC promotes keratinocyte survival,growth and migration in an autocrine manner via EPCR, epidermal growthfactor receptor and activation of ERK1/2 (17, 20).

There is a need for new approaches and therapies for treatment of, orfor minimising progression or symptoms of, disease or disorderscharacterised by keratinocyte hyperproliferation, such as psoriasis.

SUMMARY OF THE INVENTION

The invention seeks to address one or more of the above problems orlimitations, and in certain embodiments provides a method of treating anindividual for a skin disorder characterised by the presence ofhyperproliferative keratinocytes including:

-   -   providing an individual having a region of skin characterised by        the presence of hyperproliferative keratinocytes;    -   contacting the region of skin with a therapeutically effective        amount of activated protein C (APC);

thereby treating the individual for the skin disorder.

In other embodiments there is provided a method of treating anindividual for psoriasis including:

-   -   providing an individual having a psoriatic plaque;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the plaque;

thereby treating the individual for psoriasis.

In other embodiments there is provided a method of treating anindividual for acne including:

-   -   providing an individual having acne;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the acne;

thereby treating the individual for acne.

In other embodiments there is provided a method of treating anindividual for atopic dermatitis including:

-   -   providing an individual having atopic dermatitis;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the atopic dermatitis or        region of skin containing same;

thereby treating the individual for atopic dermatitis.

In other embodiments there is provided a method of treating anindividual for Pyoderma gangrenosam including:

-   -   providing an individual having Pyoderma gangrenosam;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the Pyoderma gangrenosam        or region of skin containing same;

thereby treating the individual for Pyoderma gangrenosam.

In another embodiment there is provided a method of inducing woundhealing or tissue regeneration in a region of skin having a disordercharacterised by keratinocyte hyperproliferation including:

-   -   providing an individual having a region of skin having a        disorder characterised by the presence of hyperproliferative        keratinocytes;    -   contacting the region of skin with a therapeutically effective        amount of activated protein C (APC);

thereby inducing wound healing or tissue regeneration in the skinregion.

In further embodiments there is provided a composition including atherapeutically effective amount of APC for use in minimising a skindisorder characterised by the presence of hyperproliferativekeratinocytes, or for treatment of psoriasis, acne or atopic dermatitis,in an individual.

In still further embodiments there is provided a use of APC in themanufacture of a medicament for minimising a skin disorder characterisedby the presence of hyperproliferative keratinocytes, or for treatment ofpsoriasis, acne or atopic dermatitis in an individual.

In still further embodiments there is provided a use of APC forminimising a skin disorder characterised by the presence ofhyperproliferative keratinocytes, or for treatment of psoriasis, acne oratopic dermatitis in an individual.

In still further embodiments there is provided a kit including acomposition including a therapeutically effective amount of APC andwritten instructions for use of the APC in a method of treatment of adisorder characterised by the presence of hyperproliferativekeratinocytes, or for treatment of psoriasis, acne or atopic dermatitisin an individual, the method being as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Human neonatal foreskin keratinocytes were pre-treated withAPC for 4 hr, then TNF-α and cells were incubated for 72 hours andproliferation detected by MTT assay. *P<0.05, **P<0.01 compared torelevant control. (B) Effect of APC on growth of normal mouse dermalfibroblasts (MDF), normal human dermal fibroblasts (HDF) and humanrheumatoid synovial fibroblasts (RSF, in red), measured using a crystalviolet assay (mean±SD. N=4 separate experiments. *, ‡, # p<0.01 comparedto relevant control, ANOVA. (C) RSF were treated with APC for 24 hr. p21and p27 measured by western blotting.

FIG. 2: Exogenous or endogenous APC stimulates proliferation andprevents apoptosis of cultured keratinocyte. A) Proliferation wasmeasured using an MTT assay and data expressed as % cell proliferationcompared to control. B) Proliferation of keratinocytes in response to PCsiRNA and APC treatment after 72 h as detected by MTT assay. Cellproliferation is expressed as a percentage of control. C&D)Keratinocytes were treated with PC siRNA (0.5 μm). After 48 h, cellswere used for a TUNEL (terminal dUTP nick-end labeling) assay to detectapoptotic cells (black arrows indicate apoptotic cells) (C) andquantitated by counting apoptotic cells under high power microscopy(×20) (D). Data were expressed as the average number of apoptotic cellsper field of 15 fields (mean±S.E., n=3). Graphs represent one of threeindependent experiments. Images represent one of three independentexperiments. * p<0.05, ** p<0.01. Scale bar: 40 μm.

FIG. 3: PC or EPCR siRNA treatment inhibits growth and promotesapoptosis of HUVEC.A) Proliferation rate of HUVEC in response tocontrol, PC siRNA or EPCR siRNA (500 nM) treatment in the presence orabsence of exogenous APC or PC, as detected by MTT assay. Cellproliferation is expressed as a percentage of control (mean±SD) over 72h. **P<0.01 compared to control (1st bar), ## P<0.01 compared to PCsiRNA(2nd Bar). B) Proliferation of HUVEC in response to a blocking antibodyto EPCR (RCR252) in the presence or absence of recombinant APC. Cellproliferation is expressed as a percentage of control (mean±SD) over 72h. *P<0.05 compared to control (1st bar), # P<0.05 compared to APC alone(5th bar). C-E) HUVEC were treated with control, PC siRNA or EPCR siRNA(both at 500 nM). After 36 h, transfected cells treated with recombinantAPC (1 μg/ml) for 12 h, and cells were harvested at 48 h. Activecaspase-3 was detected by immunofluorescent staining (white arrowsindicate active caspase-3 positive cells) (C) and apoptotic cells weredetected by in situ cell death detection kit (white arrows indicateapoptotic cells) (D). Images represent one of three independentexperiments. Scale bar 40 μm. E) Apoptotic cells (from D) werequantitated by counting cells co-stained with DAPI under the microscopy.Data are expressed as the average number of apoptotic cells under a highmagnification (40×) (mean±SEM n=3). *P<0.05, **P<0.01 compared tocontrol (1st bar), # P<0.05 compared to PCsiRNA treatment (2nd bar)

DETAILED DESCRIPTION OF THE EMBODIMENTS

As previously mentioned, at the time of the invention, keratinocyteswere understood to be critically involved in the pathology of a range ofskin inflammatory disorders including psoriasis. Many of the longstanding anti-inflammatory therapies were understood to beneficiallyminimise the replication of inflammatory cells, and given thatinhibition of growth and/or replication of inflammatory cells ispreferred, those compounds which had been predicted to be anti-apoptoticwere not considered for clinical use in the treatment of these disease.These are some of the reasons why, in spite of an anti-inflammatoryfunction, prior to this invention, APC had never been suggested tominimise or resolve the skin disorders characterised byhyperproliferative, hyperplastic or otherwise disregulated proliferationof keratinocytes. Indeed, prior to the invention, the published data onAPC's stimulatory effect on keratinocyte proliferation was seen to implythat APC therapy would be detrimental for inflammatory skin diseasessuch as psoriasis, acne and other skin disorders characterised bykeratinocyte hyperproliferation.

As described herein, the inventor has surprisingly found that APCinhibits apoptosis in slow growing or normal human keratinocytes orcells but has no effect on inhibiting the proliferation of abnormal orfast growing keratinocytes or cells, the latter being the mediators of awide range of inflammatory skin disorders.

From this, the inventor has recognised that in skin disorders associatedwith keratinocyte hyperproliferation, two populations of keratinocyteswith respect to response to anti-apopotic APC-mediated signals are inexistence. Specifically, those that respond to APC (i.e. that do notundergo apoptosis in response to basal levels of APC) are slow growing,normal, non inflammatory cells. Further, those that respond to APC (i.e.that do undergo apoptosis in response to basal levels of APC) are fastergrowing, abnormal inflammatory cells.

Importantly, the inventor has discovered that whilst APC can stimulateproliferation of slower growing normal cells, it has a unique selectiveability to inhibit proliferation of fast growing cells. Data is shownherein in which APC enhances proliferation and inhibits apoptosis ofslow-growing or normal human cells under basal conditions, includingendothelium and keratinocytes. However, according to the invention, APCdifferentially regulates cell proliferation depending on theinflammatory state of the cells—specifically APC inhibits growth ofTNF-α-stimulated keratinocytes and fast growing synovial fibroblastsfrom rheumatoid arthritic synovium.

From the work described herein, the inventor has conceived that,contrary to the understanding of the literature prior to the invention,the anti-inflammatory benefits of APC might be realised in patientsrequiring treatment of skin disorders characterised by keratinocytehyperproliferation, because while the anti-inflammatory effect wouldapply to the inflammatory cells, the anti-apoptotic effect should applyselectively or specifically to the keratinocytes that are noninflammatory, and not to inflammatory keratinocytes.

Further, the outcome of such treatment should be the continued normalproliferation of non inflammatory keratinocytes (arising from theanti-apoptotic function of APC) and through administration of APC, theprovision of conditions including anti inflammation, together withpossibility of allowing inflammatory, hyperproliferative keratinocytesto become apoptotic.

A. DEFINITIONS

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

“Keratinocyte” generally refers to an epidermal cell that synthesizeskeratin and other proteins and sterols. These cells constitute 95% ofthe epidermis, being formed from undifferentiated, or basal cells at thedermal-epidermal junction. Its characteristic intermediate filamentprotein is cytokeratin. In its various successive stages, keratin formsthe prickle cell layer and the granular cell layer, in which the cellsbecome flattened and slowly die to form the final layer, the stratumcorneum, which gradually exfoliates.

“Hyperproliferation” generally refers to an abnormally high rate ofproliferation of cells by rapid division. Hyperproliferation in somecontexts may arise from a cell largely existing in G₁, S or G₂ phases ofthe cell cycle. Hyperproliferation may also be referred to ashyperplasia, and hyperproliferative cells may be referred to ashyperplastic cells or tissue.

“Hyperproliferative keratinocytes” are generally keratinocytes that tendto exist in a stage of interphase of the cell cycle, such as G₁, S orG₂. These cells are unlike normal keratinocytes in that normalkeratinocytes generally exist in G₀ (Gap zero), which is either a stageseparate from interphase or an extended G₁ phase, which follows therestriction point, a cell cycle checkpoint found at the end of G₁.Hyperproliferative keratinocytes are also generally characterised byhaving a expression of inflammatory genes and in particular, genesinvolved in the NF-κB signaling pathway.

“Normal keratinocytes” are generally keratinocytes that tend to exist inG₀, or in an extended G₁ phase. Generally these keratinocytes aretypically of those that have not been stimulated with TNF-α, and they donot generally have expression of genes of the NF-κB signaling pathway.

“Activated protein C” or “APC” generally refers to a serine proteasethat functions as an anticoagulant by binding to protein S andproteolytically inactivating factors Va and VIIIa and by stimulatingfibrinolysis through neutralization of a plasminogen activatorinhibitor. Walker et al., FASEB J. 6, 2561-2567 (1992); Esmon,Arterioscler. Thromb. 12, 135-145 (1992); van Hinsbergh et al., Blood65, 444-451 (1985). Precursor protein C is produced primarily in theliver. Activation is achieved by the removal of a dodecapeptide at theN-terminus of the heavy chain of protein C. The protein C pathway isinitiated when thrombin binds to the endothelial cell surface protein,thrombomodulin, and protein C binds to the endothelial cell protein Creceptor. By inactivating factors Va and VIIIa, APC limits the amount ofthrombin formed. Esmon, Arterioscler. Thromb. 12, 135-145 (1992).

“Treating” and “treatment” generally refers to the management and careof a patient for the purpose of combating a disease, condition, ordisorder whether to eliminate the disease, condition, or disorder, orprophylactically to prevent the onset of the symptoms or complicationsof the disease, pathological condition, or disorder.

A “skin disorder characterised by the presence of hyperproliferativekeratinocytes” generally refers to a disease, condition, syndrome or thelike in which there is a predominance of hyperproliferativekeratinocytes, and in particular an abundance of hyperproliferativekeratinocytes as compared with normal tissue. The disorder may be aninflammatory disorder, and typically involving one or more of thestratum basale (stratum germinativum), stratum spinosum, stratumgranulosum, stratum lucidum and stratum corneum. Examples of skindisorders include pustular (suppurative) and non pustular (nonsuppurative) psoriasis, including the forms described herein, and acneincluding acne vulgaris or cystic acne.

A “therapeutically effective amount” generally refers to an amount ofpharmaceutical compound that provides for the desired clinical outcome.In certain embodiments, a therapeutically effective amount of APCgenerally provides an anti-apoptotic effect on normal keratinocytes andmay or may not provide an apoptotic effect on hyperproliferativekeratinocytes. In one embodiment, a therapeutically effective amount ofAPC may stop proliferation of hyperproliferative keratinocytes and/orprovide an anti-inflammatory effect. The relevant amounts can beestablished or determined according to the methods of the inventiondisclosed herein.

A “apoptosis inducing amount” generally refers to an amount of APC thatprovides for induction of apoptosis of a hyperproliferativekeratinocyte.

A “growth stimulatory amount” generally refers to an amount of APC thatprovides for induction of proliferation of non hyperproliferativekeratinocytes, and in particular, slow growing, normal, non inflammatorykeratinocytes.

An “anti-inflammatory amount” generally refers to an amount of APC thatprovides for minimisation of inflammation of a skin disordercharacterised by hyperproliferative keratinocytes.

B. METHODS OF TREATMENT

With the understanding that APC does not prevent inflammatoryhyperproliferative keratinocytes from undergoing apoptosis, the inventorhas recognised that APC can be used to provide anti-inflammatory effectsto disorders such as psoriasis and acne without aggravating or worseningthese conditions as would otherwise occur if inflammatory keratinocyteswere prevented from apoptosis or otherwise induced to proliferate orgrow, as previously thought prior to this invention. Therefore, incertain embodiments there is provided a method of treating an individualfor a skin disorder characterised by the presence of hyperproliferativekeratinocytes. The method include's:

-   -   providing an individual having a region of skin characterised by        the presence of hyperproliferative keratinocytes;    -   contacting the region of skin with a therapeutically effective        amount of activated protein C (APC);

thereby treating the individual for the skin disorder.

According to the method, the APC is delivered directly to the site orregion of the skin characterised by the presence of hyperproliferativekeratinocytes by contacting the APC or formulation containing same withthe relevant region. The site or region may be an open wound or rupturedskin surface as occurs in acne or pustular psoriasis, or it may not besubstantially ruptured, i.e. it may be more like a plaque or otherraised, hardened or fibrotic tissue. Where the site or region is open inthe sense of being ruptured or like, the APC may be applied to thesurface surrounding the rupture and/or to the exposed tissue thatnormally underlies the skin surface (for example, stratum basale(stratum germinativum), stratum spinosum, stratum granulosum, stratumlucidum).

In this context, an important finding of the inventor has been that APCcan be delivered at the required site where the skin is substantiallyunbroken, for example at a region surrounding a rupture pustule, or atan otherwise substantially unruptured skin surface. This enables localdelivery by direct contact of the APC or relevant formulation with thesite, something that is quite distinct from previous clinical systemicapplications of APC in treatment of a disseminated disease or conditionsuch as sepsis. The local administration of APC according to theinvention is advantageous and applicable to diseases which are localrather than disseminated or systemic as it limits the risk associatedwith APC delivery to the local region, and substantially minimises therisks associated with the systemic delivery of APC, for example bycontinuous infusion.

The local delivery of APC to the site of concern is a significantdeparture from the administration of APC known in the art for treatmentof other diseases and conditions. According to the methods known in theart at the time of this invention, APC is preferably administeredparenterally, most preferably intravenously, at a dose of from about 1μg/day to about 500 mg/day or from about 1 IU/kg/day to about 6000IU/kg/day for a human patient. See, e.g., U.S. Pat. Nos. 5,151,268 and5,571,786. For severe sepsis, Xigris™ is administered by continuousinfusion at a rate of from about 12 μg/kg/hr to about 30 μg/kg/hr togive a steady state plasma concentration of about 45 ng/ml APC afterabout two hours of infusion. The current invention generally is notbased on a continuous infusion by systemic administration. Rather, it isbased a local administration of APC by contacting the region of skinwith a therapeutically effective amount of APC as described herein.

The region of skin which is contacted with the therapeutically effectiveamount of APC may be inflamed, or inflammation may be in recession. Inone embodiment, the skin disorder is characterised by one or more, orall of the following processes: apoptosis, inflammation, impairedbarrier function.

In one embodiment, the region of skin is inflamed. One particularlyimportant example is a region of skin associated with chronicinflammation. This skin may have the appearance of a psoriatic plaque,and manifest in association with pustules, although the plaque may besubstantially non pustular.

The skin disorder may be selected from the group consisting ofpsoriasis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, andacne vulgaris or cystic acne.

The skin disorder may be non pustular psoriasis, for example, psoriasisvulgaris or erythrodermic psoriasis.

The skin disorder may be pustular psoriasis, for example, generalizedpustular psoriasis, pustolosis palmaris et plantaris, annula postularpsoriasis, acrodermatitis continua or impetigo herptiformis.

Typically, where the condition is psoriasis, the region of skincontacted with APC is a psoriatic plaque. Thus, in another embodimentthere is provided a method of treating an individual for psoriasis. Themethod includes:

-   -   providing an individual having a psoriatic plaque;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the plaque;

thereby treating the individual for psoriasis.

Where the condition is acne vulgaris, the region of skin contacted withAPC may include rupture and unruptured skin. Thus, in another embodimentthere is provided a method of treating an individual for acne. Themethod includes:

-   -   providing an individual having acne;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the acne;

thereby treating the individual for acne.

Where the condition is atopic dermatitis, the region of skin contactedwith APC may include rupture and unruptured skin. Thus, in anotherembodiment there is provided a method of treating an individual foratopic dermatitis. The method includes:

-   -   providing an individual having atopic dermatitis;    -   applying a composition including a therapeutically effective        amount of activated protein C (APC) to the atopic dermatitis;

thereby treating the individual for atopic dermatitis.

The atopic dermatitis may be a form of exczema selected from the groupconsisting of endogenous eczema, flexural eczema, infantile eczema, andit may also be known as “prurigo Besnier,” “neurodermitis,” or “prurigodiathesique”.

Notwithstanding the foregoing, it is understood by those skilled in theart that the dosage amount of the APC, protein C, agent that increasesthe synthesis of protein C, and/or protein C activator will vary withthe particular compound or combination of compounds employed, thedisease or condition to be treated, the severity of the disease orcondition, the type(s) of local administration, the rate of excretion ofthe compound, the duration of the treatment, the identify of any otherdrugs being administered to the animal, the age, size and species of theanimal, and like factors known in the medical arts. In general, asuitable daily dose of a compound or combination of compounds will bethat amount which is the lowest dose effective to produce a therapeuticeffect. The dosage amount, dosage form and mode of administration willbe determined by an attending physician within the scope of soundmedical judgment. Effective dosage amounts, dosage forms, and modes ofadministration for the various compounds and combination(s) of compoundscan be determined empirically and making such determinations is withinthe skill of the art.

In certain embodiments, it is important that the APC is provided so asto enable contact of APC with skin keratinocytes, as, while not wantingto be bound by hypothesis, it is believed that it is by this contactthat the APC provides selective anti-apoptotic activity (i.e. selectiveto non inflammatory non hyperproliferative cells) of the invention, andanti-inflammatory activity. Generally cells which have been contactedwith APC can be recognised by having the following characteristics,higher levels of activated matrix metalloproteinase (MMP)-2 andendothelial protein C receptor (EPCR) and lower activity of activatedMAP kinase ERK, and so contact of cells with APC, and therefore,therapeutic efficacy of treatment can be established by assessing forthese cell phenotypes.

In certain embodiments, a therapeutically effective amount of APCgenerally provides an anti-apoptotic effect on normal keratinocytes andan apoptotic effect on hyperproliferative keratinocytes. This outcomecan be assessed as follows: measuring cell survival/proliferation (MTT)assay and cell apoptosis (TUNEL assay), thereby establishing whether atherapeutically effective amount of APC has been provided. An exemplarymethod is shown further herein.

In one embodiment, a therapeutically effective amount of APC may stopproliferation of hyperproliferative keratinocytes and/or provide ananti-inflammatory effect. This outcome can be assessed as follows:

-   -   measuring cell viability/proliferation (MTT assay);    -   enzyme linked immunosorbert assay (ELISA) for inflammatory        cytokines IL-1, IL-6, IL-10, IL-17, IL-21/23 or TNF-α or    -   ELISA for EPCR (R&D Systems, Inc., MN);    -   FACS analysis with propidium iodide (PI) or PI plus annexin for        apoptosis;    -   Western blot to detect inflammatory signalling molecules such as        NF-κB;

thereby establishing whether a therapeutically effective amount of APChas been provided.

In another embodiment, the above outcomes are obtained by establishing alocal tissue concentration of APC in the region of skin from 1 ug to 100mg of APC per g of skin tissues. This can be determined by taking skinpunch biopsies under local anesthetics from the same chronic plaque. Theamount of APC may then be determined by methods known in the art. In oneexample, biopsy tissues are minced and lysed on ice. Aftercentrifugation, the clear supernatants are used to measure the PCconcentration by ELISA and APC activity by the chromogenic substrateSpectrozyme PCa assay (American Diagnostica). Enzyme activity isdetermined by measuring the increase in absorbance of the freechromophore generated per unit time at λ450 nm.

In certain embodiments, the therapeutically effective amount of APC isfrom 0.1 μg to 5000 μg of APC per cm² of the region of skin to which theAPC is applied, or from 1 μg to 2000 μg of APC per cm² of the region ofskin to which the APC is applied, or from 10 μg to 1000 μg of APC percm² of the region of skin to which the APC is applied, or from 10 μg to200 μg, or from 10 μg to 400 μg, or from 10 μg to 800 μg of APC per cm²of the region of skin to which the APC is applied

The APC may be administered once per week up to twice daily, dependingon the nature of the condition. It is generally provided for no morethan 20 weeks of consecutive days, or from no more than 6 weeks ofconsecutive days.

B.1 Methods of Treatment—Topical

Topical treatment methods, for example, using a paste, gel, cream, oil,lotion, foam, ointment or like substance are particularly useful wherethe relevant skin region is one that contains a ruptured skin surface,as this permits penetration of the APC to the relevant strata of theskin tissue where the inflammatory keratinocytes reside. However, thesetreatments may also be applied where the skin surface is substantiallyunruptured for example to a psoriatic plaque.

In one embodiment, the therapeutically effective amount of APC may befrom 0.1 to 2000 μg, preferably from 20 to 200 μg of APC per cm² of theregion of skin. A higher amount is generally preferred where the skin ismore severely affected, and generally preferred with severe chronicplaque-type psoriasis [Psoriasis Area and Severity index (PAST) ofgreater than or equal to 12, body surface area (BSA) of greater than orequal to 10] where lesions are present with ulcer, in which APC maypromote healing of ulcer. Lower amounts may be preferred where the skinis not severely affected, for example where lesions are present withoutulcer.

The concentration of APC in the formulation may be between about 10ug/ml and 1 mg/ml and the volume of composition applied to the skinregion is about 100 ul to 10 ml.

Generally where the relevant condition is psoriasis, the composition isprovided to the skin generally with a sterile surface, such as a fingeror spatula in a layer of no more than about 10 mm thickness, preferablyabout 3 mm thickness. It may then be rubbed or massaged into the skinregion and surrounding area. The application is generally from once perday to once per week, and generally no longer than 20 weeks, or nolonger than 12 weeks.

A similar application procedure and dosage regime may apply to treatmentof acne or atopic dermatitis.

In one embodiment, the APC containing composition may be applied to asolid substrate i.e. a bandage, dressing or the like, and the substratethen fixed to the relevant skin region.

B.2 Methods of Treatment—Intradermal Injection

In certain embodiments, the above outcomes are obtained by establishinga local concentration of APC at least 2 times higher than basal line.This amount of APC can be measured by measuring APC activity of skinbiopsy using ELISA and chromogenic substrate Spectrozyme PCa assay asmentioned above. Intradermal or subcutaneous injection is generallypreferred as an administration route when the stratum corneum is intactand of such nature that there is limited penetration of APC across thetopic skin layer. Generally a fine gauge needle on a (˜28-34 G) needleon a 1 ml syringe may be used. Multiple injections may be given to coverthe surface area of the skin, with ˜1 injection per cm². The amount perinjection will vary from 10 μl to 1 ml, with typical amount being 50 μl.Generally the administration is given from once per day to once perweek, and generally no longer than 20 weeks. Intradermal or subcutaneous injection can be used concurrently with topic application ofAPC. The preferred indication is psoriasis or atopic dermatitis,although other conditions characterised by keratinocytehyperproliferation may be subject to this treatment.

An exemplary embodiment of subcutaneous/intradermal administration maybe useful in severe chronic plaque-type psoriasis [Psoriasis Area andSeverity Index (PASI)≧12, body surface area (BSA)≧10] where lesions arepresent with or without ulcer. APC is administrated subcutaneously at adose of 200-200 μm depending on the size of the plaque or lesion, twicea week for 12 weeks followed by a 4-week follow-up period. The injectionis performed by 30 gauge needle with total volume of from 500 μl to 5mls, preferably 1 ml. APC is injected multiple times (intradermally orsubcutaneously) at equally-spaced sites surrounding lesion—if lesion was<10 cm², there are 4 sites and the number of sites and dose increasesproportionately with increase in lesion size. APC is dissolved in waterto make an isotonic buffered salt solution. If necessary, this treatmentcan be combined with topical treatment. APC in liquid can be applied onthe lesions, daily, continuing for same period of subcutaneoustreatment. In both studies (with or without topical treatment) the PASI,static Physician's Global Assessment (sPGA), Dermatology Life QualityIndex (DLQI), adverse events, and routine haematological and laboratoryvalues (e.g. cytokine, histological examination, PC/APC activity) can beanalysed. Expected outcomes: At week 12, >30% improvement in PASI in allpatients and >60% improvement in PAST in 50% of patients is expected.

In one embodiment, an above describe method includes the step ofcontacting the region of skin with an anti-inflammatory amount of APC.

In one embodiment, an above describe method includes the step ofcontacting the region of skin with a growth stimulatory amount of APC.

In one embodiment, an above describe method includes the step ofcontacting the region of skin with an apoptosis inducing amount of APC.

C. APC AND FORMULATIONS THEREOF

APC for use in a method described above may take the form of acomposition, or otherwise be obtained by a process, as described below.

APC may be prepared by in vitro activation of protein C purified fromplasma or prepared by recombinant DNA techniques by methods well knownin the art. See, e.g., U.S. Pat. Nos. 4,981,952, 5,151,268, 5,831,025,6,156,734, 6,268,344, and 6,395,270.

Alternatively, APC may be prepared directly by recombinant DNAtechniques. See, e.g., U.S. Pat. Nos. 4,981,952, 5,151,268, 6,156,734,6,268,344 and 6,395,270. Recombinant activated protein C may be producedby activating recombinant human protein C zymogen in vitro or by directsecretion from cells of the activated form of protein C. Protein C maybe produced in transgenic animals, transgenic plants, or a variety ofeukaryotic cells, including, for example, secretion from human kidney293 cells as a zymogen then purified and activated by techniques knownto the skilled artisan.

APC may be from any species of animal, but human APC is preferred.

Fragments and derivatives of APC may be used in the practice of theinvention, provided that they exhibit the activities described herein.See, e.g., U.S. Pat. Nos. 5,151,268, 5,453,373 and 5,516,650 and PCTapplications WO 89/12685, WO 01/56532, WO 01/59084, and WO 01/72328.

APC may be a derivative of human APC having proteolytic, amidolytic,esterolytic, and biological (anticoagulant, anti-inflammatory, orpro-fibrinolytic) activities characteristic of human APC. Examples ofprotein C derivatives are described by Gerlitz, et al., U.S. Pat. No.5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entireteachings of which are hereby incorporated by reference.

Suitable pharmaceutical compositions of APC comprise the APC and apharmaceutically-acceptable carrier. See, e.g., U.S. Pat. Nos. 6,395,270and 6,159,468 and PCT applications WO 98/48818, WO 01/56532 and WO01/72328. An APC-containing composition may generally be one that is astable lyophilized product of high purity comprising a bulking agent(such as sucrose, mannitol, trehalose, and raffinose), a salt (such assodium chloride and potassium chloride), a buffer (such as sodiumcitrate, Tris-acetate, and sodium phosphate), and APC. For example, astable lyophilized composition may comprise a weight ratio of about 1part APC, between about 7-8 parts salt, and between about 5-7 partsbulking agent. An example of such a stable lyophilized composition is:5.0 mg APC, 30 mg sucrose, 38 mg NaCl, and 7.56 mg citrate, pH 6.0, pervial.

C.1 Topically Administered Formulation

In one particularly preferred embodiment, the APC is provided in theform of a composition or formulation that is adapted for topicaladministration to a relevant skin lesion, plaque or other skin surfacethe subject of the relevant disorder according to a method describedunder Section B above. Examples of such formulations include those thatcan be applied directly to the relevant surface enabling localadministration of the APC to the relevant site. These formulationsinclude gels, oils, sprays, roll on formulations, ointments, lotions,foams and the like. In one embodiment, the APC is provided in the formof a methyl-cellulose gel and may contained stabilisers such ascarbohydrates and salts.

Skin ointment may be a combination of organic, health, beauty ormedicinal ingredients, usually in a petroleum oil base. This gives skinointment a thicker, less water-soluble formula that stays on the surfaceof the body longer so that the ingredients can work more effectively totreat a wide variety of problems. There are many natural and organicskin ointments which can be ordered from companies (such as Therapex).

Clobetasol propionate (CP) foam (0.05%) may also be used. This is anemulsion aerosol foam that has been used for the treatment ofinflammatory and pruritic manifestations of corticosteroid-responsivedermatoses in the United States and for inflammatory and pruriticmanifestations of moderate to severe atopic dermatitis in Canada (Olux-E(clobetasol propionate) foam, 0.05% Stiefel Laboratories Inc, ResearchTriangle Park, N.C. (2011).

Where the formulation is a gel, it may contain APC in an amount of10-5000 μg/g gel.

C.2 Injectable Formulation

A particularly preferred formulation of APC is the product sold by EliLilly and Co., Indianapolis, Ind., under the trademark Xigris™. Xigris™is supplied as a sterile, lyophilized powder for intravenous infusion.The 5 mg vials of Xigris™ contain 5.3 mg/vial of human recombinant APC,31.8 mg/vial sucrose, 40.3 mg/vial NaCl, and 10.9 mg/vial sodiumcitrate, and the 20 mg vials of Xigris™ contain 20.8 mg/vial of humanrecombinant APC, 124.9 mg/vial sucrose, 158.1 mg/vial NaCl, and 42.9mg/vial sodium citrate. The vials are reconstituted with Sterile Waterfor Injection, USP, to give a concentration of about 2 mg/ml APC, andthis diluted APC is then added to 0.9% Sodium Chloride Injection to givea concentration of from about 100 to about 5000 μg/ml APC foradministration to a patient. This is a particularly preferredformulation for administration of APC by subcutaneous injectiontechniques as described under Section B above.

Whether administered topically or by sub cutaneous injection, in certainembodiments, the relevant formulation may contain protein C as analternative to, or in addition to APC. For instance, an effective amountof protein C can be administered which will be activated in vivo by theendogenous protein C pathway to produce APC. See, e.g., U.S. Pat. No.5,151,268 and PCT application WO 93/09807. As noted above, protein C canbe purified from plasma or can be made by recombinant DNA techniques.See, e.g., U.S. Pat. Nos. 4,959,318, 4,981,952, 5,093,117, 5,151,268,5,571,786, 6,156,734, 6,268,344, and 6,395,270. Suitable pharmaceuticalcompositions comprising protein C are known (see, e.g., U.S. Pat. Nos.5,151,268 and 5,571,786).

Endogenous production of APC can also be increased by administering anamount of an agent that increases the synthesis of protein C in theanimal. See, e.g., PCT application WO 93/09807. Suitable agents includeanabolic steroids (e.g., danazolol). See, e.g., PCT application WO93/09807.

In certain embodiments, endogenous production of APC can be increased byadministering an amount of a protein C activator effective to cause theproduction of APC in vivo from endogenously synthesized protein C and/orfrom co-administered protein C. See, e.g., PCT application WO 93/09807.A protein C activator is any compound that causes or increases thegeneration of APC. Suitable protein C activators include thrombin,α-thrombin, active site acylated thrombin, thrombin analogs and mutants(e.g., thrombin E192Q and thrombin K52E), solublethrombin-thrombomodulin complexes, agents that would prevent clearanceor decay of thrombin-thrombomodulin complexes, agents that enhance thesynthesis or delay the clearance of thrombomodulin, a venom (such asProtac or Russel Viper venom), factor Xa, plasmin, trypsin, and anyother venom, enzyme or compound capable of causing or increasing thegeneration of APC from protein C. See, e.g., PCT application WO93/09807. Preferred protein C activators are thrombin and active siteacylated thrombin.

In some embodiments, APC may be administered with another agent forcontrolling one or more or inflammation, cell proliferation andapoptosis. One particularly preferred agent is an anti-IL-17 antibody,in particular Ixekizumab, which showed significant improvements in skindisease severity scores compared with placebo in a Phase II Study inPatients With Chronic Plaque Psoriasis (NEJM, 2012). Other examples ofagents for controlling inflammation include TNF-α inhibitors andanti-inflammatory cytokines and biopharmaceuticals.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

EXAMPLES Example 1 Pre-Clinical Trial Establishing the SelectiveAnti-Apoptotic Activity of APC on Slow Growing, Non InflammatoryKeratinocytes and Psoriasis Skin Keratinocytes

Six patients with active chronic plaque psoriasis and 6 normalindividuals are recruited. They have not received any treatment for atleast 4 weeks prior to sampling. Two 6-mm punch biopsies are taken underlocal anesthetic from the same chronic plaque. Keratinocytes areisolated as we described previously (20).

Normal keratinocytes are treated with a mixture of cytokines [IL-1α (10ng/ml), IL-6 (5 ng/ml), TNF-α (5 ng/ml), IL-17A (10 ng/ml)] to induce apsoriatic phenotype. APC is added to keratinocytes at 1, 10 μg/ml andtreated for 24, 48 and 72 hrs. Cell proliferation/survival is examinedusing MIT assay, Brdu proliferation assay. Cell apoptosis is examined byTUNEL assay, Flow cytometry (propidium iodide (PI) or PI plusannexin-V). Cytokine production and four selected psoriasis-associatedgenes TNF, DEFB4, CAMP, PI3 are detected by RT-real time PCR and ELISA.The activation and expression of apoptotic signal molecules capase-3, 8and 9 and MAK kinase ERK are detected by western blot.

The results will show that APC induces the apoptosis of, and slows thegrowth of psoriastic keratinocytes while it stimulates the growth andsurvival of the normal keratinocytes. APC also reduces the levels ofinflammatory cytokines IL-1, TNF-α, IL-17 and IL-6 andpsoriasis-associated molecules TNF, DEFB4, CAMP, PI3. In the normalcontrol cells that are treated with inflammatory mediators, cell growthis enhanced and the addition of APC reverses this effect. Overall, theresults will clearly show that APC can not only inhibit the inflammationassociated with psoriatic keratinocytes, but it also reduces thecharacteristic excessive proliferation associated with these cells.

Example 2 A Phase 2 Pilot Trial with Subcutaneous APC Over 12 Weeks

Patient selection: 5 Patients with severe chronic plaque-type psoriasis[Psoriasis Area and Severity Index (PASI)≧12, body surface area(BSA)≧10] are enrolled. Primary inclusion criteria includes patients 18to 70 years of age who have had stable plaque psoriasis for at least 6months. Primary exclusion criteria includes: i) patients with non-plaqueor drug-induced psoriasis; ii) the use of biologicals such as rituximab,abatacept, infliximab, adalimumab, cyclosporine, or mycophenolic acidand etanercept or anakinra within 28 days prior to study iii) patientswho have received anti-psoriatic treatment, including any phototherapy,during 8 weeks preceding the study and treatment with any standardtopical therapy for psoriasis other than with bland emollients during 4weeks preceding the study; iv) the use of topical therapy during thestudy is limited to class III to VII glucocorticoids on the scalp,axillae, and groin only; v) evidence of any active or recent infections,or a history of malignancy or other autoimmune disease, pregnant womenare also exclusion criteria.

Treatment: Patients are administered with 400 ug APC/plaquesubcutaneously twice a week for up to 12 weeks or when plaques resolve.APC is injected evenly around (as previously described) the peripheryand under psoriatic plaques and vehicle alone (placebo) is injectedsimilarly into control plaques symmetrically localized on the other bodyside. Patients are then followed up for an additional 4 weeks after thefinal treatment.

Measurement: The PAST, static Physician's Global Assessment (sPGA),Dermatology Life Quality Index (DLQI), adverse events, and routinehaematological and laboratory values (e.g. cytokine, histologicalexamination, PC/APC activity) are analysed. Safety considerationsinclude bleeding risk, headache, fatigue, infection and allergy.Relevant cell and biochemistry events include white blood cell,neutrophil, and platelet counts; coagulation activity, PC/APC levels andantibody formation to APC in serum. A 4 mm biopsy is taken to performhistological examination of PC/APC and EPCR, and the evaluation of themain psoriatic parameters such as acanthosis, hyperkeratosis, andparakeratosis, the mitotic activity of the epidermis, papillary edema,dilation and tortuosity of capillaries, and neutrophils in the dermis,the stratum spinosum, as well as in the horny layer, respectively.

Statistical analysis: Baseline values and values at weeks duringtreatment, and 4 weeks after treatment are compared at a significancelevel of 0.05, using a 1-sided 2-sample t-test.

Results and Discussion: APC treatment will be shown to be safe and welltolerated and patients will show at least 60% resolution of psoriasislesions, with 3 of 5 patients showing complete resolution of the plaque.APC's effect shall be sustained during the 4 week follow-up. Allpatients will show improved PASI, sPGA, DLQI and with no adverse effectsand no APC antibodies formed in response to the treatment. Significantreductions in the thickness of epidermis and inflammatory cells in theskin can be observed, and an increase in keratinocyte expression ofEPCR, however, circulating PC/APC levels are not affected. Overall, theresults will show that APC is safe, well tolerated and highlyefficacious for psoriasis plaques.

Example 3 A Phase 2 Pilot Trial with Subcutaneous aPC for Acne Vulgarisin 10 Patients

Patient selection: 10 Patients with acne vulgaris are enrolled. Primaryinclusion criteria includes: patients 18 to 30 years of age who have hadbilateral facial acne for at least 6 months. Primary exclusion criteriaincludes evidence of any active or recent infections, or a history ofmalignancy or other autoimmune disease, pregnant women.

Treatment: Patients are administered with 200 ug APC in gel form once aday for 12 weeks or when acne resolves. The patients are provided withtwo tubes of gel marked L and R and advised to apply 2 cm of gel,squeezed from the tube, to the designated affected area (L on left sideof face and R on right side of face) and rub in evenly. Patients arethen followed up for an additional 4 weeks after the final treatment.

Measurement: Photographs are taken before treatment and every 4 weeksand acne area calculated using computer-assisted image analysis. Adverseevents, and routine haematological and laboratory values (e.g. cytokine,histological examination, PC/APC activity) are analysed. Safetyincluding bleeding risk, headache, fatigue, infection, allergy aremonitored. Relevant cell and biochemistry events include white bloodcell, neutrophil, and platelet counts; coagulation activity, PC/APClevels and antibody formation to APC in serum.

Statistical analysis: Baseline values and values at weeks duringtreatment, and 4 weeks after treatment were compared at a significancelevel of 0.05, using a 1-sided 2-sample t-test.

Results and Discussion: APC treatment will be shown to be safe and welltolerated and patients will show at least 50% resolution of the acne.APC's effect will be sustained during the 4 week follow-up. There willbe no adverse effects and no APC antibodies formed in response to thetreatment. There should be significant reductions in the thickness ofepidermis and inflammatory cells in the skin, an increase inkeratinocyte expression of EPCR, however, circulating PC/APC levels arenot affected. Overall, our results will show that APC is safe, welltolerated and highly efficacious for acne vulgaris.

Example 4 Gel Formulation and Therapeutic Application

This example offers a non-sterile, low bioburden, preserved, sodiumcarboxymethylcellulose-based topical gel, containing the activeingredient activated protein C and the following inactive ingredients:carboxymethylcellulose sodium, glacial acetic acid, 1-lysinehydrochloride, m-cresol, methylparaben, propylparaben, sodium acetatetrihydrate, sodium chloride, and water for injection. Each gram of Gelcontains 100 ug activated protein C.

Example 5 Gel Formulation and Therapeutic Application

Carbopol®*Ultrez 30 polymer (Lubrizol Advanced Materials, Inc) offersgood viscosity in combination with xanthan gum in the presence of 1%salicylic acid and 5% of an electrolyte containing extract, in this coldprocess formulation at pH 4. Glucam™*E 20 humectant, along withglycerin, imparts humectancy. Below is the formula.

INCI Name, Trade Name Weight % Function A. 1. Deionized Wafer 79.15Diluent 2. Disodium EDTA, 0.05 Chelating Protachem NA2 Agent 3.Carbomer, 0.80 Rheology Carbopol ®* Ultrez 30 Polymer Modifier B. 4.Glycerin, 2.00 Humectant Glycerin, 99.7% USP 5. Methyl Gtoceth-20, 1.00Humectant Glucamm ™* E-20 Humectant 6. Xanthan Gum, 0.25 ThickenerKeltrol ® CG C. 7. Sodium Hydroxide (18% Solution) 1.75 Neutralizer D.8. Butylene Glycol 6.00 Solubilizer 9. Alcohol, 2.00 Solubilizer EthylAlcohol 10. Salicylic Acid 1.00 Anti-Acne Agent E. 11. PEG-40Hydrogenated Castor Oil, 0.25 Solubilizer Cremophor ® CO-40 12. LaurylLactate, 0.25 Emollient Schercemol ™* LL Ester F. 13. Phenoxyethanol(and) 0.50 Preservative Ethylhexylglycerin, Euxyl ® PE9010 14. Water,Glycerin 5.00 Anti-irritant

Procedure:

1. Combine PART D ingredients and mix until Salicylic Acid Crystals arefully dissolved. Set aside for later addition.

2. Dissolve Disodium EDTA in water. When Disodium EDTA is fullydissolved, set mixing speed @400-500 rpm and slowly disperseCarbopol®*Ultrez 30 polymer into water. Continue mixing until polymer isfully hydrated.

3. Premix PART B ingredients into a uniform slurry. Add to PART A.Increase mixing speed as needed to maintain uniformity.

4. Add PART C to PART AB and increase mixing speed as needed to maintainuniformity. Continue mixing for 10 minutes.

5. Add PART D to batch and adjust mixing speed as needed to reduce anysplashing due to viscosity change. Mix until uniform.

6. Premix PART E ingredients and add to batch. Mix until uniform.

7. Add PART F ingredients individually with good mixing betweenadditions.

APC is added to an amount of 10-5000 gel or 0.01-0.05 w/w %

This gel can be applied directly to lesion areas.

Example 6 Spray on Formulations and Therapeutic Applications

APC was solubilised in an isotonic sterile solution as described abovefor APC by injection. Spray delivery was achieved by drawing up thecontents of the vial into a 2.5 ml syringe using a drawing needle. Asterilised spray nozzle was fitted to the end of the syringe (in placeof the needle) and the treatment (APC or saline) was evenly sprayed ontothe plaque in order to cover the entire affected surface. Thus, nopropellant was required to obtain atomisation, merely the pressureapplied to the syringe. Application was repeated as necessary,preferably once a day. This technique is easy to perform and repeat in aconsistent manner providing an even coverage of treatment.

Example 7

Techniques for assessment of anti-apoptotic effect on normalkeratinocytes and apoptotic effect on hyperproliferative keratinocytes.

According to the invention, a therapeutically effective amount of APCgenerally provides an anti-apoptotic effect on normal keratinocytes andan apoptotic effect on hyperproliferative keratinocytes. This outcomecan be assessed in vitro or in situ as follows:

In Vitro Method 1.

Untreated active psoriatic skin is removed using a biopsy punch andkeratinocytes are isolated. Briefly, cells (3000 cells/well) are seededin 96 well-plates to a final volume of 200 μl, then incubated for 4hours to allow cells to attach. Cells are then treated with APC at 0.1,1 and 10 μg/ml for 72 hrs.

1) Cell survival/growth rate by MTT assay: In this assay, mitochondrialdehydrogenases of viable cells cleave the tetrazolium ring, yieldingpurple MTT formazan crystals which are insoluble in aqueous solutions.The crystals can be dissolved in acidified isopropanol. The resultingpurple solution is spectrophotometrically measured. An increase in cellnumber results in an increase in the amount of MTT formazan formed andan increase in absorbance. Three hours prior to the completion of thetreatment, 10 μl of 5 mg/ml MTT is added to cells. After a furtherincubation 3 hours, the MTT solution is removed and replaced by 100 μlDMSO. The optical density of each well is determined at a wavelength of570 nm with a reference wavelength of 630 nm. The viability of cells isdirectly related to OD. APC reduced the viability in a dose-dependentmanner.

2) Apoptotic rate detection was by TUNEL assay, FACS analysis withpropidium iodide (PI) or PI plus annexin-V) and active caspase activity.

TUNEL assay is performed using an in situ cell death detection kitaccording to manufacturer's instructions (Roche Diagnostics AustraliaPty. Ltd., NSW, Australia). Briefly, cells are permeabilised with 0.1%Triton X-100 in freshly prepared 0.1% sodium citrate, and incubated withterminal deoxynucleotidyl transferase in the presence offluorescein-labeled dUTP (60 min at 37° C.). TUNEL positive cells werevisualized using an anti-fluorescein peroxidase (POD) conjugatedantibody and POD substrate reaction. Sections were counterstained withDAPI. The frequency of apoptotic cells was determined by a blindedinvestigator by counting TUNEL positive cells and total cell numberunder a high magnification view (40×) and calculating the percentage ofTUNEL positive cells.

FACS analysis: Keratinocytes are trypsinized and washed with FACSwashing buffer (PBS with 5% FCS). A 200 μl cell suspension is incubatedwith propidium iodide (PI) or PI plus annexin-V or conjugatedantibodies, and subsequently detected by a flow cytometer for cellcycles/cell apoptosis and protein expression. Data is analyzed usingFlowJo software.

Active caspase activity is detected by western blot. Followingtreatment, keratinocytes are washed three times with PBS and lysisbuffer (0.15 M NaCl, 0.01 mM PMSF, 1% NP-40, 0.02 M Tris, 6 M urea/H₂O)supplemented with protease inhibitor and phosphate inhibitor (Roche,Indianapolis, Ind., USA) was added. Cell lysates are centrifuged at10,000 g for 15 minutes and supernatants are separated by 10%sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS-PAGE)and transferred to a PDVF membrane. Anti-human caspase-3, 8 and 9antibodies are detected. Immunoreactivity is detected using the ECLdetection system (Amersham, Piscataway, N.J.). Anti-human β-actinantibody was included to normalize against unequal loading.

In all apoptosis assays APC promotes apoptosis of fast-growingkeratinocytes.

In Vitro Method 2.

The aim is to determine whether keratinocytes from APC-treated plaquesare more prone to apoptosis than placebo-treated cells. APC is deliveredto psoriatic plaques (˜5-50 cm² in size) using 2 methods: i) topicalinjection (100 ng-1 mg); ii) spraying a solution (10 ng-1 mg), usingsterilised spray nozzle on end of 0.5 ml syringe). Active psoriatic skintreated with either APC or placebo are removed using biopsy punches andkeratinocytes are isolated. Cells are immediately measured for apoptosisusing FACS analysis, as described above. Results indicated that thereare significantly higher levels of apoptotic cells from APC-treatedskin, compared to placebo-treated skin.

In Situ Method

in situ cell apoptosis: The aim is to determine whether keratinocytesfrom APC-treated plaques are more prone to apoptosis thanplacebo-treated cells. APC is delivered to psoriatic plaques (˜5-50 sqcm in size) using 2 methods: i) topical injection (100 ng-1 mg); ii)spraying a solution (10 ng-1 mg), using sterilised spray nozzle on endof 0.5 ml syringe). Active psoriatic skin treated with either APC orplacebo is removed using biopsy punches. Skin is fixed and cellapoptosis is detected using an in situ cell death detection kitaccording to manufacturer's instructions (Roche Diagnostics AustraliaPty. Ltd., NSW, Australia). Briefly, cells are permeabilised with 0.1%Triton X-100 in freshly prepared 0.1% sodium citrate, and incubated withterminal deoxynucleotidyl transferase in the presence offluorescein-labeled dUTP (60 min at 37° C.). TUNEL positive cells arevisualized using an anti-fluorescein peroxidase (POD) conjugatedantibody and POD substrate reaction. Sections are counterstained withDAPI. The frequency of apoptotic cells is determined by a blindedinvestigator by counting TUNEL positive cells and total cell numberunder a high magnification view (40×) and calculating the percentage ofTUNEL positive cells. Skin treated with APC has significantly higherlevels of TUNEL-positive apoptotic cells compared to placebo-treatedskin.

Mouse Model of Psoriasis

The psoriasis xenograft SCID mouse model (82 mice required) is performedas previously described (Raychaudhuri, 2001 Br. J. Dermatol). Briefly,human psoriatic plaques (1 cm²) are grafted onto the back of an˜8-week-old SCID mice after removing a full-thickness skin sample. Thepsoriatic grafts are accepted in 3-4 weeks and the psoriasis-likedisease is maintained for at least 10 weeks. APC is delivered afterpsoriasis development using 2 methods: i) intradermal injection (10ng-100 ug); ii) spraying a solution (10 ng-100 ug), using a sterilisedspray nozzle on the end of 0.5 ml syringe) on affected skin (8/group) 3times/week for up to 8 weeks. Physiological saline is used as placebocontrol. Punch biopsies (2 mm), taken on day 0 (before treatment) andday 28 (after treatment), are snap frozen and processed forimmunostaining and histology to determine epidermal thickness, rete peglength (an indicator of epidermal thickness) and inflammatory cellularinfiltrates. On day 28, skin is collected to measure inflammatorymediators, differentiation markers and junctional proteins. APCtreatment reduces the thickness of the skin and the number ofinflammatory cells, particularly the neutrophils and T cells.Inflammatory cytokines in APC-treated skin are reduced, particularlytumour necrosis factor-α and junction-associated proteins are increased,especially zona occludens-1 claudins, occludins, and JAM-A, compared toplacebo.

Cell proliferation using BrdU incorporation: To achieve labelling ofcells undergoing DNA synthesis, adult mice receive one injection of BrdU(100 mg/kg) 2 h before euthanasia, and mouse skins are collected andfixed with 10% formalin. Proliferative cells are detected byimmunostaining with anti-BrdU antibody. APC-treated cells exhibitsignificantly less BrdU incorporation indicating lower proliferativecapacity.

Example 8 A Phase 2 Pilot Trial with Subcutaneous APC Over One Month inFive Patients

Patients

Patients 18 to 70 years of age who had stable plaque psoriasis for atleast 6 months, had received or were candidates for phototherapy orsystemic psoriasis therapy.

Exclusions:

Patients with non-plaque or drug-induced psoriasis.

Patients who had received anti-psoriatic treatment, including anyphototherapy, during 8 weeks preceding the study and treatment with anystandard topical therapy for psoriasis other than with bland emollientsduring 4 wk preceding the study. The use of topical therapy during thestudy would be limited to class III to VII glucocorticoids on the scalp,axillae, and groin only.

Patients with clinical evidence for infection during 3 weeks before thestudy, and patients with a history of cancer.

Study Design

A phase 2 pilot trial with subcutaneous APC (100 ug) weekly for onemonth in five patients.

APC Administration Protocol.

Five otherwise healthy, male patients with chronic plaque psoriasis arechosen for treatment with APC. The drug is administered weekly over aperiod of 4 weeks in a dosage of 100 ug.

APC is injected subcutaneously directly under psoriatic plaques andvehicle alone (placebo) is injected under a control plaque symmetricallylocalized on the other body side. A control plaque comparable to theother two is selected for study with neither injection

Reporting

Descriptive Analysis (Baseline)

The baseline demographic (age, weight, BMI, ethnicity) and diseasecharacteristics (duration of psoriasis, percentage of body area affectedby psoriasis, mean PASI score, the presence of marked or severepsoriasis), as assessed by means of the static physician's globalassessment, prior topical treatment, prior phototherapy, prior systemictherapy, prior biologic therapy.

Efficacy and Safety Evaluations

Efficacy from day 1 (before therapy) up to 3 d after the end of therapy(day 31) by photo documentation and/or 20 mHz ultrasound.

Three different plaques (including the site of APC and vehicleadministration) are monitored in detail in each patient to determine thereduction in the echo-poor band (representing both epidermal acanthosisand the infiltrate in the upper dermis) the percentage improvement inthe psoriasis area and severity index (PASI) of each patient.

Skin biopsies (4-mm punch biopsies) 4 day before APC at one week and atday 24 (end of therapy) for histological and immune-histochemicalexamination as well as mRNA.

Histology and Immunohistochemistry

The histological investigations include the evaluation of the mainpsoriatic parameters such as acanthosis, hyperkeratosis, andparakeratosis, the mitotic activity of the epidermis, papillary edema,dilation and tortuosity of capillaries, and neutrophils in the dermis,the stratum spinosum, as well as in the horny layer, respectively

Immunohistochemical Investigations

Safety

Adverse events, serious adverse events, Routine hematologic andlaboratory values, Antibody formation to APC.

Side-effects and the pruritus

Delayed type of hypersensitivity reaction (DTH)

REFERENCES

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1. A method of treating an individual for a skin disorder characterisedby the presence of hyperproliferative keratinocytes including: providingan individual having a region of skin characterised by the presence ofhyperproliferative keratinocytes; contacting the region of skin with atherapeutically effective amount of activated protein C (APC); therebytreating the individual for the skin disorder.
 2. The method of claim 1wherein the therapeutically effective amount of APC minimisesinflammation in the region of skin.
 3. The method of claim 1 wherein thetherapeutically effective amount of APC minimises apoptosis in theregion of skin.
 4. The method of claim 1 wherein the therapeuticallyeffective amount of APC does not inhibit the growth of normalkeratinocytes, or induces proliferation of normal keratinocytes.
 5. Themethod of claim 1 wherein the therapeutically effective amount of APC isfrom 1 ug to 5 mg of APC per cm² of the region of skin.
 6. The method ofclaim 1 wherein the APC is provided for contact with the region of skinin the form of a gel, cream, ointment, spray, lotion or like formulationadapted for contact with the skin surface.
 7. The method of claim 1wherein the APC is provided for contact with the region of skin in theform of a composition adapted for subcutaneous injection.
 8. The methodof claim 1 wherein the skin disorder is characterised by one or more ofthe following processes: apoptosis, inflammation, impaired barrierfunction.
 9. The method of claim 1 wherein the skin disorder is selectedfrom the group consisting of psoriasis, dermatitis herpetiformis,pemphigus vulgaris, and vitiligo.
 10. The method of claim 9 wherein theskin disorder is non pustular psoriasis.
 11. The method of claim 10wherein the psoriasis is psoriasis vulgaris or erythrodermic psoriasis.12. The method of claim 9 wherein the skin disorder is pustularpsoriasis.
 13. The method of claim 12 wherein the psoriasis isgeneralized pustular psoriasis, pustolosis palmaris et plantaris, annulapostular psoriasis, acrodermatitis continua or impetigo herptiformis.14. The method of claim 1 wherein the region of skin contacted with APCis a psoriatic plaque.
 15. The method of claim 1 including the furtherstep of providing an anti-inflammatory agent to the individual. 16-19.(canceled)